After treatment of neurotoxin 11, a component part of the venom of the Middle Asian cobra Naja naju oxiana, with acetoxysuccinimide all five possible e-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacety1)-neurotoxins I1 and the hexa(trifluoroacety1)-neurotoxin 11, which were studied by circular dichroism (CD), 'H and I9F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five &-amino groups) in the hexa(trifluoroacety1)-neurotoxin 11 NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the "F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin I1 I9F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.Among the various components of snake venom, the neurotoxins are endowed not only with high potency but with an exceptional specificity of action. The most extensively studied members of this group, the postsynaptic neurotoxins, block transmission of the nerve impulse by binding to the acetylcholine receptor in the membrane [l -41. The present work is a continuation of our spectroscopic studies of postsynaptic neurotoxins [5 -71 aiming at their dynamic molecular structure in solution, this being of crucial importance for understanding toxin-receptor interactions. Up to now the most informative results have been obtained from the 'H NMR study of neurotoxin 11, the primary structure of which is given in Fig.1. In the above-mentioned work, chemical modification was used as an aid in the spectral assignments; however, in the present study it is used as an equal partner of NMR, considerably broadening its scope.We prepared a neurotoxin I1 derivative whose amino groups were all trifluoroacetylated and conAbbreviations. CD, circular dichroism, NMR, nuclear magnetic resonance; ESR, electron spin resonance; spin probe, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl.--~ ducted the 19F NMR study of this compound, the signals of which were assigned with the aid of a series of selectively acetylated-trifluoroacetylated derivatives. As a result, intramolecular interactions and the extent of solvent exposure of trifluoroacetyl groups were discovered; this, together with the earlier data [6,7], yields a better understanding of the folding of the neurotoxin backbone.,.-.
We studied the ability of four non-conjugated alpha7-subunit fragments of the nicotinic acetylcholine receptor to induce an immune response and to protect memory in olfactory bulbectomized mice which demonstrate abnormalities similar to Alzheimer's disease (AD). Vaccination only with the alpha7-subunit fragment 173-193 was shown to rescue spatial memory, to restore the level of alpha7 acetylcholine receptors in the cortex, and to prevent an increase in the amyloid-beta (Abeta) level in brain tissue in these animals. Antibodies against the peptide 173-193 were revealed in blood serum and cerebrospinal liquid in the bulbectomized mice. Passive immunization with mouse blood sera containing antibodies to the peptide 173-193 also restored memory in bulbectomized animals. The observed positive effect of both active and passive immunization with the fragment of alpha7-subunit on memory of bulbectomized mice provides a new insight into an anti-AD drug design.
A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin I1 from the venom of Middle-Asian cobra Nuju nuju oxiunu has been performed in 2H20 and H20 solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in HzO solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C (2)H histidine resonances.The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.Protein neurotoxins from snake venoms play an important role in the elucidation of the mechanism of reception of biologically active compounds. Neurotoxins that are capable of blocking nervous impulse transmission by highly specific interaction with acetyl choline receptor of postsynaptic membrane are now objects of intensive investigation; they also provide a tool for isolating the respective receptor proteins and studying their properties [l].Chemical modification of amino acid residues in neurotoxins showed that the native three-dimensional structure is one of the most important factors for the biological activity of these compounds [2]. The conformation of neurotoxins has been studied by optical rotatory dispersion and circular dichroism data and analysis of variability of the neurotoxin primary structure. Invaluable information both on general features of the protein globule and on characteristics of particular amino acid residues may be inferred from the NMR studies. This method, along with some other spectroscopic techniques, has been used by us in conformational studies of toxins from the venom of Naju nuju oxiana (Middle-Asian cobra) [13]. The present paper deals with the NMR study of one of them, namely 'short' postsynaptically acting neurotoxin I1 which is composed of 61 amino acid residues and cross-linked by four disulfide bridges. The amino acid sequence (Fig.1) of this protein has been established in two laboratories [14,15]. The proton NMR spectra of neurotoxin I1 have been recorded in H20 and 2H20 with 100-MHz and 300-MHz instruments over a wide range of temperature and pH ; deuterium exchange studies have been performed for amide protons and his...
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